Sandbox 16

Please do NOT make changes to this sandbox. Sandboxes 10-30 are currently reserved by Professor Jaswal (Dr. J) at Amherst College.

Alpha-lytic protease
Alpha-lytic protease (αLP) is a 198 residue extracellular bacterial serine protease produced by Lysobacter enzymogenes. The three-dimensional fold of αlp puts it in the same class as cymotrypsin, trypsin and other digestive serine proteases despite only modest sequence homology. However, unlike its thermodynamically stable homologs, αLP is stabilized by a large unfolding activation barrier. This kinetic stability optimizes the native state to survive under the harsh, proteolytic conditions in which it operates. Since the native state is less stable than both an intermediate and a completely unfolded state, αLP requires a Pro region to facilitate folding by stabilizing the folding transition state as well as the native state. After folding, the pro region is proteolytically cleaved, leaving an active αLP kinetically trapped.

Structure
The β-strands (yellow), α-helices (pink) and loops (white) constitute the secondary structure. The tertiary structure contains the β-barrel motif and disulfide bonds that are characteristic of the chymotrypsin family as well as an active site containing the "catalytic triad" - His 57, Asp 102, and Ser 195 - that is responsible for proteolysis. The preference for αLP to cleave substrates on the C-terminal side of small hydrophobic residues, such as Alanine and Valine is mostly due to three residues in the S1 pocket consisting of Met 190, Met 213, and Val 218. The N to C coloring and important structural regions are shown here, with the molecule colored dark blue at the N-terminus and progressing to red at the C-terminus. The Domain Bridge is the only covalent linkage between the two domains and has been shown to modulate the unfolding rate. Interestingly, the domain bridge, cis-proline turn, and C-terminal β-hairpin are found in kinetically stable proteases but not in their thermodynamically stable family members, like chymotrypsin and trypsin. Thus it is not surprising that these regions play an integral role in the concerted unfolding of αLP. Compared to trypsin and other thermodynamic homologs, where relatively small unfolding events at the transition state can expose the buried interface, αLP has highly cooperative substructures that protect the domain interface from solvent.

Folding
In contrast to its mammalian homologs like trypsin and chymotrypsin, αLP is synthesized with a 166 residue N-terminal Pro region that plays an obligatory role in the proper folding of its 198 residue protease domain. The Pro region overcomes the barrier to folding by providing a catalyzed pathway in which the transition state to folding is lowered by 18.2 kcal/mol. The product of this folding is not active αLP but an inhibitory complex, N*P. The release of active αLP requires the removal of the Pro region via proteolysis, which occurs naturally. This leaves the native αLP, a metastable state with a large barrier to unfolding (t1/2~1.2 years). Below shows the free energy diagrams summarizing the difference between the folding landscape of a typical thermodynamically stable protein (left) and that of the kinetically stable αLP (right). The free-energy diagram of αLP folding is shown with (dotted blue line) and without (solid black line) its Pro region (P). In the absence of its Pro region, unfolded αLP (U) spontaneously folds to a partially folded intermediate (I), which progresses at a very slow rate (t1/2 ~1800 years) to N through a very high transition state (TS).

Rainbow ALp TextToBeDisplayed --Student 17:24, 28 June 2010 (IDT)Paul Cohen